Stabilized receptor polypeptides and uses thereof

ABSTRACT

The present invention provides stabilized activin IIB receptor polypeptides and proteins capable of binding and inhibiting the activities of activin A, myostatin, or GDF-11. The present invention also provides polynucleotides, vectors and host cells capable of producing the stabilized polypeptides and proteins. Compositions and methods for treating muscle-wasting diseases and metabolic disorders are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.13/775,756, filed Feb. 25, 2013, which claims benefit of U.S. Ser. No.12/626,375 (U.S. Pat. No. 8,410,043), filed Nov. 25, 2009, which claimsthe benefit of U.S. Provisional Application Ser. No. 61/200,250, filedon Nov. 26, 2008, and U.S. Provisional Application Ser. No. 61/259,060,filed on Nov. 6, 2009, the entire disclosures of which are relied uponand incorporated by reference herein.

REFERENCE TO THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 21, 2016, is named32735US_CRF_sequencelisting.txt, and is 84 kilobytes in size.

TECHNICAL FIELD OF THE INVENTION

The technical field of this invention relates to transforming growthfactor-β (TGF-β) family members and soluble TGF-β receptors withimproved properties, as well as methods of modulating the activities ofTGF-β family members for the treatment of various disorders.

BACKGROUND OF THE INVENTION

The transforming growth factor β (TGF-β) family of proteins includes thetransforming growth factors-β (TGF-β), activins, bone morphogenicproteins (BMP), nerve growth factors (NGFs), brain-derived neurotrophicfactor (BDNF), and growth/differentiation factors (GDFs). These familymembers are involved in the regulation of a wide range of biologicalprocesses including cell proliferation, differentiation, and otherfunctions.

Growth/differentiation factor 8 (GDF-8), also referred to as myostatin,is a TGF-ß family member expressed for the most part in the cells ofdeveloping and adult skeletal muscle tissue. Myostatin appears to playan essential role in negatively controlling skeletal muscle growth(McPherron et al., Nature (London) 387, 83-90 (1997), Zimmers et al.,Science 296:1486-1488 (2002)). Antagonizing myostatin has been shown toincrease lean muscle mass in animals.

Another member of the TGF-β family of proteins is a relatedgrowth/differentiation factor, growth/differentiation factor 11(GDF-11). GDF-11 has approximately 90% sequence identity to the aminoacid sequence of myostatin. GDF-11 has a role in the axial patterning indeveloping animals (Oh et al., Genes Dev 11:1812-26 (1997)), and alsoappears to play a role in skeletal muscle development and growth.

Activins A, B and AB are the homodimers and heterdimer respectively oftwo polypeptide chains, βA and βB (Vale et al., Nature 321, 776-779(1986), Ling et al., Nature 321, 779-782 (1986)). Activins wereoriginally discovered as gonadal peptides involved in the regulation offollicle stimulating hormone synthesis, and are now believed to beinvolved in the regulation of a number of biological activities. ActivinA is a predominant form of activin.

Activin, myostatin, GDF-11 and other members of the TGF-β superfamilybind and signal through a combination of activin type II and activintype IIB receptors, both of which are transmembrane serine/threoninekinases (Harrison et al., J. Biol. Chem. 279, 28036-28044 (2004)).Cross-linking studies have determined that myostatin is capable ofbinding the activin type II receptors ActRIIA and ActRIIB in vitro (Leeet al., PNAS USA 98:9306-11 (2001)). There is also evidence that GDF-11binds to both ActRIIA and ActRIIB (Oh et al., Genes Dev 16:2749-54(2002)).

TGF-β protein expression is known to be associated with a variety ofdiseases and disorders. Therefore, therapeutic molecules capable ofantagonizing several TGF-β proteins simultaneously may be particularlyeffective for treating these diseases and disorders.

Production of therapeutic proteins on a commercial scale requiresproteins that can be efficiently expressed and purified withoutdisruption of the integrity of the protein. Manufacturability can bedescribed as the ability to express and purify a protein in asufficiently efficient manner to allow for cost-effective production ofthe protein. In a commercial setting, manufacturability must bedetermined for each potential therapeutic protein. Although proteinexpression and purification processes can be optimized for a protein,manufacturability appears to be a function of the intrinsic propertiesof the protein as well. The present invention provides biologicallyactive therapeutic proteins having improved manufacturabilityproperties, capable of effectively antagonizing TGF-β proteins.

SUMMARY OF THE INVENTION

The present invention provides isolated proteins comprising stabilizedhuman activin receptor JIB (designated svActRIIB) polypeptides capableof binding and inhibiting the activities of activin, GDF-11 andmyostatin, and characterized by improved manufacturability properties.The stabilized ActRIIB polypeptides are characterized by having aminoacid substitutions at both positions 28 and 44 with respect to SEQ IDNO: 2.

In one embodiment, the isolated protein comprises a polypeptide havingthe sequence set forth in SEQ ID NO: 2, except for a single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected from Wor Y, and the substitution at position 44 is T. In another embodiment,the polypeptide has the sequence set forth in amino acids 19 through 134of SEQ ID NO: 2, except for a single amino acid substitution at position28, and a single amino acid substitution at position 44, wherein thesubstitution at position 28 is selected from W or Y, and thesubstitution at position 44 is T. In another embodiment, the polypeptidehas the sequence set forth in amino acids 23 through 134 of SEQ ID NO:2, except for a single amino acid substitution at position 28, and asingle amino acid substitution at position 44, wherein the substitutionat position 28 is selected from W or Y, and the substitution at position44 is T. In another embodiment, the polypeptide has the sequence setforth in amino acids 25 through 134 of SEQ ID NO: 2, except for a singleamino acid substitution at position 28, and a single amino acidsubstitution at position 44, wherein the substitution at position 28 isselected from W or Y, and the substitution at position 44 is T. Inanother embodiment, the polypeptide has an amino acid sequence with atleast 80%, 85%, 90%, 95%, 98% or 99% identity to any of the polypeptidesabove, wherein the polypeptide has single amino acid substitution atposition 28, and a single amino acid substitution at position 44,wherein the substitution at position 28 is selected from W or Y, and thesubstitution at position 44 is T, and wherein the polypeptide is capableof binding myostatin, activin A, or GDF-11. In one embodiment, thesubstitution of the above polypeptides at position 28 is W and thesubstitution at position 44 is T, wherein the polypeptide is capable ofbinding myostatin, activin A, or GDF-11.

In another embodiment, the isolated protein comprises a stabilizedactivin IIB receptor polypeptide, wherein the polypeptide has thesequence set forth in the group consisting of SEQ ID NO: 4, 6, 12 and14. In another embodiment the protein comprises a polypeptide having atleast 80% sequence identity to SEQ ID NO: 4, 6, 12 or 14, wherein thepolypeptide has a W or Y at position 28 and a T at position 44, andwherein the polypeptide is capable of binding myostatin, activin A, orGDF-11. In another embodiment the protein comprises a polypeptide havingat least 90% sequence identity to SEQ ID NO: 4, 6, 12 or 14, wherein thepolypeptide has a W or Y at position 28 and a T at position 44, andwherein the polypeptide is capable of binding myostatin, activin A, orGDF-11. In another embodiment, the protein comprises a polypeptidehaving at least 95% sequence identity to SEQ ID NO: 4, 6, 12, or 14,wherein the polypeptide has a W or Y at position 28 and a T at position44, and wherein the polypeptide is capable of binding myostatin, activinA, or GDF-11. In one embodiment, the substitution at position 28 is Wand the substitution at position 44 is T, wherein the polypeptide iscapable of binding myostatin, activin A, or GDF-11.

In a further embodiment the svActRIIB protein further comprises aheterologous protein. In one embodiment, the heterologous protein is anFc domain. In a further embodiment, the Fc domain is a human IgG Fcdomain. In a further embodiment the heterologous protein is attached bya linker or a hinge linker peptide. In one embodiment, the linker orhinge linker is selected from group consisting of the amino acidsequences set forth in the group consisting of SEQ ID NO: 25, 27, 38,40, 42, 44, 45, 46, 48, 49 and 50. In a further embodiment the hingelinkers set forth in SEQ ID NO: 27, 38, 40, 42, 44, 45, or 46 link thehuman IgG2 Fc (SEQ ID NO: 22) to an svActRIIB polypeptide. In anotherembodiment, the hinge linkers set forth in SEQ ID NO: 48, 49, or 50 linkthe human IgG1 Fc (SEQ ID NO: 23) or the modified IgG1 Fc (SEQ ID NO:47) to an svActRIIB polypeptide.

In a further embodiment, the protein comprises a polypeptide having thesequence set forth in the group consisting of SEQ ID NO: 8, 10, 16 and18. In another embodiment the protein comprises a polypeptide having atleast 80% sequence identity to SEQ ID NO: 8, 10, 16 or 18, wherein thepolypeptide has a W or Y at position 28 and a T at position 44, andwherein the polypeptide is capable of binding myostatin, activin A, orGDF-11. In another embodiment the protein comprises a polypeptide havingat least 90% sequence identity to SEQ ID NO: 8, 10, 16 or 18, whereinthe polypeptide has a W or Y at position 28 and a T at position 44, andwherein the polypeptide is capable of binding myostatin, activin A, orGDF-11. In another embodiment, the protein comprises a polypeptidehaving at least 95% sequence identity to SEQ ID NO: 8, 10, 16, or 18,wherein the polypeptide has a W or Y at position 28 and a T at position44, and wherein the polypeptide is capable of binding myostatin, activinA, or GDF-11. In a further embodiment, the substitution of the abovepolypeptides at position 28 is W and the substitution at position 44 isT, wherein the polypeptide is capable of binding myostatin, activin A,or GDF-11.

In a further embodiment, the protein comprises the polypeptides recitedabove, wherein the amino acid residue at position 64 is alanine.

In another aspect the present invention provides an isolated nucleicacid molecule comprising a polynucleotide encoding a stabilized ActRIIBpolypeptide. In one embodiment, the polynucleotide encodes thepolypeptide sequence set forth in SEQ ID NO: 2, except for a singleamino acid substitution at position 28, and a single amino acidsubstitution at position 44, wherein the substitution at position 28 isselected from W or Y, and the substitution at position 44 is T. Inanother embodiment, the polynucleotide encodes the polypeptide havingthe sequence set forth in amino acids 19 through 134 of SEQ ID NO: 2,except for a single amino acid substitution at position 28, and a singleamino acid substitution at position 44, wherein the substitution atposition 28 is selected from W or Y, and the substitution at position 44is T. In another embodiment, the polynucleotide encodes the polypeptidehaving the sequence set forth in amino acids 23 through 134 of SEQ IDNO: 2, except for a single amino acid substitution at position 28, and asingle amino acid substitution at position 44, wherein the substitutionat position 28 is selected from W or Y, and the substitution at position44 is T. In another embodiment, the polynucleotide encodes thepolypeptide having the sequence set forth in amino acids 25 through 134of SEQ ID NO: 2, except for a single amino acid substitution at position28, and a single amino acid substitution at position 44, wherein thesubstitution at position 28 is selected from W or Y, and thesubstitution at position 44 is T. In another embodiment, thepolynucleotide encodes the a polypeptide having an amino acid sequenceat least 80%, 85%, 90%, 95%, 98% or 99% identity to any one of thepolypeptides above, wherein the polypeptide has single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected from Wor Y, and the substitution at position 44 is T, and wherein thepolypeptide is capable of binding myostatin, activin A, or GDF-11. Inone embodiment, the above polynucleotides encode a polypeptide whereinthe substitution at position 28 is W and the substitution at position 44is T, wherein the polypeptide is capable of binding myostatin, activinA, or GDF-11.

In one embodiment, the nucleic acid molecule comprises a polynucleotideencoding a polypeptide having the sequence set forth in the groupconsisting of SEQ ID NO: 4, 6, 12 and 14. In another embodiment, thenucleic acid molecule comprises a polynucleotide encoding a polypeptidehaving at least 80% sequence identity to SEQ ID NO: 4, 6, 12 or 14,wherein the polypeptide has a W or Y at position 28 and a T at position44, and wherein the polypeptide is capable of binding myostatin, activinA, or GDF-11. In another embodiment, the nucleic acid molecule comprisesa polynucleotide encoding a polypeptide having at least 90% sequenceidentity to SEQ ID NO: 4, 6, 12 or 14, wherein the polypeptide has a Wor Y at position 28 and a T at position 44, and wherein the polypeptideis capable of binding myostatin, activin A, or GDF-11. In anotherembodiment, the nucleic acid molecule comprises a polynucleotideencoding a polypeptide having at least 95% sequence identity to SEQ IDNO: 4, 6, 12 or 14, wherein the polypeptide has a W or Y at position 28and a T at position 44, and wherein the polypeptide is capable ofbinding myostatin, activin A, or GDF-11. In one embodiment, the abovepolynucleotides encode a polypeptide wherein the substitution atposition 28 is W and the substitution at position 44 is T, wherein thepolypeptide is capable of binding myostatin, activin A, or GDF-11.

In another embodiment, the nucleic acid molecule comprises apolynucleotide having a sequence selected from the group consisting ofSEQ ID NO: 3, 5, 11 and 13, or its complement.

In another embodiment, the isolated nucleic acid molecule comprises thepolynucleotides set forth above, and further comprises a polynucleotideencoding at least one heterologous protein. In one embodiment, thenucleic acid molecule comprises a polynucleotide encoding a polypeptidehaving the sequence set forth in the group consisting of SEQ ID NO: 8,10, 16 and 18. In another embodiment, the nucleic acid moleculecomprises a polynucleotide encoding a polypeptide having at least 80%sequence identity to SEQ ID NO: 8, 10, 16 or 18, wherein the polypeptidehas a W or Y at position 28 and a T at position 44, and wherein thepolypeptide is capable of binding myostatin, activin A, or GDF-11. Inanother embodiment, the nucleic acid molecule comprises a polynucleotideencoding a polypeptide having at least 90% sequence identity to SEQ IDNO: 8, 10, 16 or 18, wherein the polypeptide has a W or Y at position 28and a T at position 44, and wherein the polypeptide is capable ofbinding myostatin, activin A, or GDF-11. In another embodiment, thenucleic acid molecule comprises a polynucleotide encoding a polypeptidehaving at least 95% sequence identity to SEQ ID NO: 8, 10, 16 or 18,wherein the polypeptide has a W or Y at position 28 and a T at position44, and wherein the polypeptide is capable of binding myostatin, activinA, or GDF-11. In one embodiment, the above polynucleotides encode apolypeptide wherein the substitution at position 28 is W and thesubstitution at position S44 is T, wherein the encoded polypeptide iscapable of binding myostatin, activin A or GDF-11. In a furtherembodiment, the nucleic acid molecule comprises a polynucleotide havinga sequence selected from the group consisting of SEQ ID NO: 7, 9, 15 and17, or its complement.

In another embodiment, the nucleic acid molecule further comprisespolynucleotides encoding the linkers and hinge linkers set forth in thegroup consisting of SEQ ID NO: 25, 27, 38, 40, 42, 44, 45, 46, 48, 49and 50.

In a further embodiment, the nucleic acid molecule further comprises atranscriptional or translational regulatory sequence. In another aspecta recombinant vector comprising a polynucleotide encoding a stabilizedActRIIB protein or polypeptide is provided. In another aspect, hostcells comprising the recombinant vectors are provided, and methods ofproducing the stabilized ActRIIB proteins and polypeptides are providedby culturing the host cells under conditions promoting expression of theproteins or polypeptides.

The present invention further provides a composition containing at leastone stabilized ActRIIB polypeptide or protein of the present invention.In one embodiment, the composition is a pharmaceutical compositioncontaining the stabilized ActRIIB polypeptide or protein in admixturewith a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method of reducing orblocking myostatin, activin A or GDF-11 activity by administering thesvActRIIB proteins and polypeptides, or pharmaceutical compositionscontaining these, to a subject in need of such treatment.

In another aspect, the invention provides a method of increasing leanmuscle mass or increasing the ratio of lean muscle mass to fat mass in asubject in need of such treatment by administering an effective amountof the composition or pharmaceutical composition containing svActRIIBproteins or polypeptides to the subject.

In another aspect, the invention provides a method of treating orpreventing a muscle wasting disease in a subject suffering from such adisorder by administering a therapeutic composition containing ansvActRIIB polypeptide or protein to the subject. The muscle wastingdisease includes, but is not limited to, the following conditions:cancer cachexia, muscular dystrophy, amyotrophic lateral sclerosis,congestive obstructive pulmonary disease, chronic heart failure,chemical cachexia, cachexia from HIV/AIDS, renal failure, uremia,rheumatoid arthritis, age-related sarcopenia, age-related frailty, organatrophy, carpal tunnel syndrome, androgen deprivation, andmuscle-wasting due to inactivity from prolonged bed rest, spinal chordinjury, stroke, bone fracture, burns, aging, insulin resistance, andother disorders. The muscle wasting may also result from weightlessnessdue to space flight.

In another aspect, the present invention provides a method of treatingconditions in which activin is overexpressed in a subject in need ofsuch treatment, by administering an effective amount of a therapeuticcomposition containing svActRIIB proteins or polypeptides to thesubject. In one embodiment, the disease is cancer. In another aspect,the present invention provides a method of treating a metabolic disordercomprising administering a therapeutic composition containing svActRIIBproteins or polypeptides to a subject in need of such treatment, whereinthe metabolic disorder is selected from bone loss, diabetes, obesity,impaired glucose tolerance, hyperglycemia, and metabolic syndrome. Inanother aspect, the present invention provides a method of gene therapyfor treating muscle wasting or metabolic or activin-related disorderscomprising administering a vector encoding an svActRIIB polypeptide orprotein of the present invention to a subject in need thereof, whereinthe vector is capable of expressing the svActRIIB protein or polypeptidein the subject.

In another aspect, the present invention provides a method of detectingand quantitating myostatin, activin, or GDF-11 by using any of thesvActRIIB proteins or polypeptides as capture or binding agents in anynumber of assays.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a comparison between ActRIIB-Fc (E28W) and svActRIIB-Fc(E28W, S44T) on an SEC column. svActRIIB-Fc (E28W, S44T) shows a singlepeak compared with ActRIIB-Fc (E28W), which shows three peaks.

FIG. 2 shows the increase in body mass over a 14 day period in 10C57B1/6 mice administered a single dose of 10 mg/kg svActRIIB-Fc (E28W,S44T) compared with 10 mice administered 10 mg/kg of PBS.

FIG. 3 shows the dose-related change in lean body mass over time forC57B1/6 receiving a single dose of 0.3 mg/kg, 3 mg/kg, 10 mg/kg, and 30mg/kg of svActRIIB-Fc (E28W, S44T).

DETAILED DESCRIPTION

The present invention provides an isolated protein comprising astabilized human activin IIB receptor (svActRIIB) polypeptide. Theprotein and polypeptide of the invention are characterized by theirability to bind to at least one of three TGF-β proteins, myostatin(GDF-8), activin A, or GDF-11, to inhibit the activities of at least oneof these proteins, and to have improved manufacturability propertiescompared with other ActRIIB soluble receptors. The stabilized humanactivin IIB receptor polypeptide is characterized by amino acidsubstitutions at both positions E28 and S44 with reference to theextracellular domain of ActRIIB, as set forth in SEQ ID NO: 2. In oneembodiment, a stabilized human activin IIB receptor polypeptide can havea further substitution of alanine at position 64 with respect to SEQ IDNO: 2.

As used herein the term “TGF-β family members” or “TGF-β proteins”refers to the structurally related growth factors of the transforminggrowth factor family including activins, and growth and differentiationfactor (GDF) proteins (Kingsley et al. Genes Dev. 8: 133-146 (1994),McPherron et al., Growth factors and cytokines in health and disease,Vol. 1B, D. LeRoith and C. Bondy. ed., JAI Press Inc., Greenwich, Conn.,USA: pp 357-393).

GDF-8, also referred to as myostatin, is a negative regulator ofskeletal muscle tissue (McPherron et al. PNAS USA 94:12457-12461(1997)). Myostatin is synthesized as an inactive protein approximately375 amino acids in length, having GenBank Accession No: AAB86694 (SEQ IDNO: 35) for human. The precursor protein is activated by proteolyticcleavage at a tetrabasic processing site to produce an N-terminalinactive prodomain and an approximately 109 amino acid C-terminalprotein which dimerizes to form a homodimer of about 25 kDa. Thishomodimer is the mature, biologically active protein (Zimmers et al.,Science 296, 1486 (2002)).

As used herein, the term “prodomain” or “propeptide” refers to theinactive N-terminal protein which is cleaved off to release the activeC-terminal protein. As used herein the term “myostatin” or “maturemyostatin” refers to the mature, biologically active C-terminalpolypeptide, in monomer, dimer or other form, as well as biologicallyactive fragments or related polypeptides including allelic variants,splice variants, and fusion peptides and polypeptides. The maturemyostatin has been reported to have 100% sequence identity among manyspecies including human, mouse, chicken, porcine, turkey, and rat (Leeet al., PNAS 98, 9306 (2001)).

As used herein GDF-11 refers to the BMP (bone morphogenic protein)having Swissprot accession number 095390 (SEQ ID NO: 36), as well asvariants and species homologs of that protein. GDF-11 is involved in theregulation of anterior/posterior patterning of the axial skeleton(McPherron et al, Nature Genet. 22 (93): 260-264 (1999); Gamer et al,Dev. Biol. 208 (1), 222-232 (1999)) but postnatal functions are unknown.

Activin A is the homodimer of the polypeptide chains PA. As used hereinthe term “activin A” refers to the activin protein having GenBankAccession No: NM_002192 (SEQ ID NO: 34). Activins A, B, and AB are thehomodimers and heterodimer respectively of two polypeptide chains, βAand βB. As used herein, “activin” refers to activin A, B, and AB, aswell as variants and species homologs of that protein.

Receptor Polypeptides

As used herein, the term activin type II B receptors (ActRIIB) refers tohuman activin receptors having accession number NP_001097 or variantsthereof, such as those having the arginine at position 64 substitutedwith alanine. The term soluble ActRIIB (wild type) refers to theextracellular domain of ActRIIB, amino acids 1 to 134 (with signalsequence), or amino acids 19 through 134 of SEQ ID NO: 2 (without signalsequence).

Stabilized Receptor Polypeptides

The present invention provides an isolated protein comprising astabilized ActIIB receptor polypeptide (referred herein as “svActRIIBpolypeptide”). As used herein the term “svActRIIB protein” refers to aprotein comprising a stabilized ActRIIB polypeptide. As used herein theterm “isolated” refers to a protein or polypeptide molecule purified tosome degree from endogenous material. These polypeptides and proteinsare characterized as having the ability to bind and inhibit the activityof any one of activin A, myostatin, or GDF-11, in addition to havingimproved manufacturability characteristics.

The stabilized ActRIIB polypeptide is characterized by having an aminoacid substitution at both position 28 and 44 with respect to SEQ ID NO:2. For consistency, the amino acid positions on the stabilized ActRIIBpolypeptides and proteins are always referred to with respect to thepositions in SEQ ID NO: 2, regardless of whether the polypeptide ismature or truncated. As used herein, the term “mature” refers to apolypeptide or peptide without its signal sequence. As used herein, theterm “truncated” refers to polypeptides having N terminal amino acids orC terminal amino acids removed.

In one embodiment, the isolated stabilized activin IIB receptorpolypeptide (svActRIIB) has the polypeptide sequence set forth in SEQ IDNO: 2, except for a single amino acid substitution at position 28, and asingle amino acid substitution at position 44, wherein the substitutionat position 28 is selected from W or Y, and the substitution at position44 is T. In another embodiment, the polypeptide has the sequence setforth in amino acids 19 through 134 of SEQ ID NO: 2, except for a singleamino acid substitution at position 28, and a single amino acidsubstitution at position 44, wherein the substitution at position 28 isselected from W or Y, and the substitution at position 44 is T. Inanother embodiment, the polypeptide has the sequence set forth in aminoacids 23 through 134 of SEQ ID NO: 2, except for a single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected from Wor Y, and the substitution at position 44 is T. In another embodiment,the polypeptide has the sequence set forth in amino acids 25 through 134of SEQ ID NO: 2, except for a single amino acid substitution at position28, and a single amino acid substitution at position 44, wherein thesubstitution at position 28 is selected from W or Y, and thesubstitution at position 44 is T. In another embodiment, the polypeptidehas an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identity to any one of the polypeptides above, wherein thepolypeptide has single amino acid substitution at position 28, and asingle amino acid substitution at position 44, wherein the substitutionat position 28 is selected from W or Y, and the substitution at position44 is T, and wherein the polypeptide is capable of binding myostatin,activin A, or GDF-11. In one embodiment, the substitution of the abovepolypeptides at position 28 is W, and the substitution at position 44 isT, wherein the polypeptide is capable of binding myostatin, activin A,or GDF-11.

In one embodiment, the svActRIIB polypeptide includes a signal sequence,for example, SEQ ID NO: 4, 8, 12, and 16. However, various signalpeptides can be used in the preparation of the polypeptides of theinstant application. The signal peptides can have the sequence set forthin amino acids 1 to 19 of SEQ ID NO: 4, for example, or the signalsequences set forth in SEQ ID NO: 31 and 32. Any other signal peptidesuseful for expressing svActRIIB polypeptides may be used. In otherembodiments, the signal sequence is removed, leaving the mature peptide.Examples of svActRIIB polypeptides lacking a signal sequence includes,for example, SEQ ID NO: 6, 10, 14 and 18.

In one embodiment, the protein comprises a stabilized activin IIBreceptor polypeptide, wherein the polypeptide is selected from the groupconsisting of polypeptides having the sequence set forth in the groupconsisting of SEQ ID NO: 4, 6, 12 and 14. These polypeptides representamino acids 25 to 134 of SEQ ID NO: 2, wherein the polypeptide hassingle amino acid substitution at position 28, and a single amino acidsubstitution at position 44, wherein the substitution at position 28 isselected from W or Y, and the substitution at position 44 is T, andwherein the polypeptide is capable of binding myostatin, activin A, orGDF-11, with and without a signal sequence different from that shown inSEQ ID NO: 2. In another embodiment the protein comprises a polypeptidehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO: 4, 6, 12 or 14, wherein the polypeptide has a Wor Y at position 28 and a T at position 44, and wherein the polypeptideis capable of binding myostatin, activin A, or GDF-11. In oneembodiment, the substitution at position 28 is W and the substitution atposition 44 is T, wherein the polypeptide is capable of bindingmyostatin, activin A or GDF-11.

In a further embodiment the svActRIIB protein further comprises aheterologous protein. In one embodiment, the heterologous protein is anFc domain. In a further embodiment, the Fc domain is a human IgG Fcdomain. In one embodiment, the protein comprises a polypeptide havingthe sequence set forth in the group consisting of SEQ ID NO: 8, 10, 16and 18. In another embodiment, the protein comprises a polypeptidehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO: 8, 10, 16 or 18, wherein the polypeptide has a Wor Y at position 28 and a T at position 44, and wherein the polypeptideis capable of binding myostatin, activin A, or GDF-11. In oneembodiment, the substitution at position 28 is W and the substitution atposition 44 is T, wherein the polypeptide is capable of bindingmyostatin, activin A or GDF-11.

In a further embodiment, the protein comprises the any one of thepolypeptides described above, wherein the amino acid residue at position64 is alanine.

In another embodiment, the term svActRIIB polypeptide and proteinencompasses proteins comprising fragments of SEQ ID NO: 2, 4, 6, 12 and14, including N and C terminal truncations, wherein position 28 is W orY, and position 44 is T, and wherein the polypeptide is capable ofbinding myostatin, activin A or GDF-11.

As used herein the term “derivative” of the svActRIIB polypeptide refersto the attachment of at least one additional chemical moiety, or atleast one additional polypeptide to form covalent or aggregateconjugates such as glycosyl groups, lipids, acetyl groups, or C-terminalor N-terminal fusion polypeptides, conjugation to PEG molecules, andother modifications which are described more fully below. StabilizedActRIIB receptor polypeptides can also include additional modificationsand derivatives, including modifications to the C and N termini whicharise from processing due to expression in various cell types such asmammalian cells, E. coli, yeasts and other recombinant host cells.

The svActRIIB proteins of the present invention may further compriseheterologous polypeptides attached to the svActRIIB polypeptide eitherdirectly or through a linker sequence to form a fusion protein. As usedherein the term “fusion protein” refers to a protein having aheterologous polypeptide attached via recombinant DNA techniques.Heterologous polypeptides include but are not limited to Fcpolypeptides, his tags, and leucine zipper domains to promoteoligomerization and further stabilization of the stabilized ActRIIBpolypeptides as described in, for example, WO 00/29581, which is hereinincorporated by reference. In one embodiment, the heterologouspolypeptide is an Fc polypeptide or domain. In one embodiment, the Fcdomain is selected from a human IgG1 Fc (SEQ ID NO: 23), modified IgG1Fc (SEQ ID NO: 47), IgG2 Fc (SEQ ID NO: 22), and IgG4 Fc (SEQ ID NO: 24)domain. The svActRIIB protein can further comprise all or a portion ofthe hinge sequence of the IgG1 (SEQ ID NO: 29), IgG2 (SEQ ID NO: 28), orIgG4 (SEQ ID NO: 30). Exemplary svActRIIB polypeptides are selected frompolypeptides consisting of the sequences as set forth in SEQ ID NO: 8,10, 16 and 18, as well as those polypeptides having substantialsimilarity to these sequences, wherein the substitutions at positions 28and 44 are retained. As used herein, “substantial similarity” refers tosequences that are at least 80% identical, 85% identical, 90% identical,95% identical, 96% identical, 97% identical, 98% identical, 99%identical to any of SEQ ID NO: 8, 10, 16, and 18, wherein thepolypeptides retain W or Y at position 28 and T at position 44, andwherein the polypeptide is capable of binding myostatin, activin A orGDF-11. In one embodiment, the substitution at position 28 is W and thesubstitution at position 44 is T, wherein the polypeptide is capable ofbinding myostatin, activin A or GDF-11.

The svActRIIB polypeptide can optionally further comprise a “linker”sequence. Linkers serve primarily as a spacer between a polypeptide anda second heterologous polypeptide or other type of fusion or between twoor more stabilized ActRIIB polypeptides. In one embodiment, the linkeris made up of amino acids linked together by peptide bonds, preferablyfrom 1 to 20 amino acids linked by peptide bonds, wherein the aminoacids are selected from the 20 naturally occurring amino acids. One ormore of these amino acids may be glycosylated, as is understood by thoseof skill in the art. In one embodiment, the 1 to 20 amino acids may beselected from glycine, alanine, proline, asparagine, glutamine, andlysine. In one embodiment, a linker is made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine.Exemplary linkers are polyglycines (particularly (Gly)₅, (Gly)₈,poly(Gly-Ala), and polyalanines. One exemplary suitable linker as shownin the Examples below is (Gly)₄Ser (SEQ ID NO: 25). In a furtherembodiment, svActRIIB can comprise a “hinge linker”, that is a linkersequence provided adjacent to a hinge region or a partial hinge regionof an IgG, as exemplified in SEQ ID NO: 27. Hinge sequences includeIgG2Fc (SEQ ID NO: 28), IgG1Fc (SEQ ID NO: 29), and IgG4Fc (SEQ ID NO:30).

Hinge linker sequences may also be designed to improve manufacturabilityand stability of the svActRIIB-Fc proteins. In one embodiment, the hingelinkers of SEQ ID NO: 27, 38, 40, 42, 44, 45, and 46 are designed toimprove manufacturability with the IgG2 Fc (SEQ ID NO: 22) when attachedto svActRIIB polypeptides. In one embodiment, the hinge linker sequencesis designed to improve manufacturability when attaching svActRIIBpolypeptides to a human IgG1 Fc (SEQ ID NO: 23) or a modified human IgG1Fc (SEQ ID NO: 47), for example, the hinge linkers having SEQ ID NO: 48,SEQ ID NO: 49 and SEQ ID NO: 50. The improved manufacturability of thesepolypeptides is described below in Example 4.

Linkers may also be non-peptide linkers. For example, alkyl linkers suchas —NH—(CH₂)s-C(O)—, wherein s=2-20 can be used. These alkyl linkers mayfurther be substituted by any non-sterically hindering group such aslower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂,phenyl, etc.

The svActRIIB polypeptides disclosed herein can also be attached to anon-polypeptide molecule for the purpose of conferring desiredproperties such as reducing degradation and/or increasing half-life,reducing toxicity, reducing immunogenicity, and/or increasing thebiological activity of the svActRIIB polypeptides. Exemplary moleculesinclude but are not limited to linear polymers such as polyethyleneglycol (PEG), polylysine, a dextran; a lipid; a cholesterol group (suchas a steroid); a carbohydrate, or an oligosaccharide molecule.

The svActRIIB proteins and polypeptides have improved manufacturabilityproperties when compared to other ActRIIB soluble polypeptides. As usedherein, the term “manufacturability” refers to the stability of aparticular protein during recombinant expression and purification ofthat protein. Manufacturability is believed to be due to the intrinsicproperties of the molecule under conditions of expression andpurification. Examples of improved manufacturability characteristics areset forth in the Examples below and include uniform glycosylation of aprotein (Example 2), increased cell titer, growth and protein expressionduring recombinant production of the protein (Example 1), improvedpurification properties (Example 2), and improved stability at low pH(Example 2). The svActRIIB proteins and polypeptides of the presentinvention demonstrate the improved manufacturability, along withretention of in vitro and in vivo activity (Examples 2 and 3), comparedwith other soluble ActRIIB polypeptides. Further, additional hingelinker sequences may confer additional manufacturability benefits, asshown in Example 4 below.

As used herein, the term a “svActRIIB polypeptide activity” or “abiological activity of a soluble ActRIIB polypeptide” refers to one ormore in vitro or in vivo activities of the svActRIIB polypeptidesincluding but not limited to those demonstrated in the Example below.Activities of the svActRIIB polypeptides include, but are not limitedto, the ability to bind to myostatin or activin A or GDF-11, and theability to inhibit or neutralize an activity of myostatin or activin Aor GDF-11. As used herein, the term “capable of binding” to myostatin,activin A, or GDF-11 refers to binding measured by methods known in theart, such as the KinExA™ method shown in the Examples below. In vitroinhibition of myostatin, activin A, or GDF-11 can be measured using, forexample, the pMARE C2C12 cell-based assay described in the Examplesbelow. In vivo activity, demonstrated in Example 3 below, isdemonstrated by increased lean muscle mass in mouse models. In vivoactivities of the svActRIIB polypeptides and proteins include but arenot limited to increasing body weight, increasing lean muscle mass, andincreasing the ratio of lean muscle to fat mass. Therapeutic activitiesfurther include reducing or preventing cachexia caused by certain typesof tumors, preventing the growth of certain types of tumors, andincreasing survival of certain animal models. Further discussion of thesvActRIIB protein and polypeptide activities is provided below.

In another aspect, the present invention provides an isolated nucleicacid molecule comprising a polynucleotide encoding an svActRIIBpolypeptide of the present invention. As used herein the term “isolated”refers to nucleic acid molecules purified to some degree from endogenousmaterial.

In one embodiment, the polynucleotide encodes a polypeptide having thesequence set forth in SEQ ID NO: 2, except for a single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected from Wor Y, and the substitution at position 44 is T. In another embodiment,the polynucleotide encodes a polypeptide having the sequence set forthin amino acids 19 through 134 of SEQ ID NO: 2, except for a single aminoacid substitution at position 28, and a single amino acid substitutionat position 44, wherein the substitution at position 28 is selected fromW or Y, and the substitution at position 44 is T. In another embodiment,the polynucleotide encodes a polypeptide having the sequence set forthin amino acids 23 through 134 of SEQ ID NO: 2, except for a single aminoacid substitution at position 28, and a single amino acid substitutionat position 44, wherein the substitution at position 28 is selected fromW or Y, and the substitution at position 44 is T. In another embodiment,the polynucleotide encodes a polypeptide having the sequence set forthin amino acids 25 through 134 of SEQ ID NO: 2, except for a single aminoacid substitution at position 28, and a single amino acid substitutionat position 44, wherein the substitution at position 28 is selected fromW or Y, and the substitution at position 44 is T. In another embodiment,the polynucleotide encodes the a polypeptide having an amino acidsequence at least 80%, 85%, 90%, 95%, 98% or 99% identity to any one ofthe polypeptides above, wherein the polypeptide has single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected from Wor Y, and the substitution at position 44 is T, and wherein thepolypeptide is capable of binding myostatin, activin A, or GDF-11. Inone embodiment, the polynucleotide of the above embodiments encodes apolypeptide wherein the substitution at position 28 is W and thesubstitution at position 44 is T.

In one embodiment, the isolated nucleic acid molecule of the presentinvention comprises a polynucleotide encoding a polypeptide having thesequence set forth in the group consisting of SEQ ID NO: 4, 6, 12, and14. In another embodiment, the nucleic acid comprises a polynucleotideencoding a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%sequence identity to SEQ ID NO: 4, 6, 12 or 14, wherein the polypeptidehas a W or Y at position 28 and a T at position 44, and wherein thepolypeptide is capable of binding activin A, GDF-11, or myostatin. Inone embodiment, the polynucleotide of the above embodiments encodes apolypeptide wherein the substitution at position 28 is W and thesubstitution at position 44 is T, and wherein the polypeptide is capableof binding activin A, GDF-11 or myostatin.

In another embodiment, the isolated nucleic acid molecule furthercomprises a polynucleotide encoding at least one heterologous protein.In one embodiment, the heterologous protein is an Fc domain, in afurther embodiment, the Fc domain is a human IgG Fc domain. In anotherembodiment, the nucleic acid molecule further comprises polynucleotidesencoding the linkers and hinge linkers set forth in SEQ ID NO: 25, 27,38, 40, 42, 44, 45, 46, 48, 49 or 50. In a further embodiment, suchpolynucleotides have sequences selected from the group consisting of SEQID NO: 26, 37, 39, 41, and 43.

In one embodiment, the nucleic acid molecule comprises a polynucleotideencoding a polypeptide consisting of the sequence set forth in the groupconsisting of SEQ ID NO: 8, 10, 16 and 18. In another embodiment, thenucleic acid comprises a polynucleotide encoding a polypeptide having atleast 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to the groupconsisting of SEQ ID NO: 8, 10, 16 and 18, wherein the polypeptide has aW or Y at position 28 and a T at position 44, and wherein thepolypeptide is capable of binding activin A, GDF-11, or myostatin. Inone embodiment, the polynucleotide of the above embodiments encodes apolypeptide wherein the substitution at position 28 is W and thesubstitution at position 44 is T, and wherein the polypeptide is capableof binding myostatin, activin A or GDF-11.

In one embodiment, the isolated nucleic acid molecule comprises apolynucleotide having the sequence selected from the group consisting ofSEQ ID NO: 3, 5, 11 or 13, or its complement. In another embodiment, theisolated nucleic acid molecule comprises a polynucleotide having thesequence selected from the group consisting of the sequence SEQ ID NO:7, 9, 15 and 17, or its complement. In a further embodiment the isolatednucleic acid molecule hybridizes under stringent or moderate conditionswith SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17 wherein the encodedpolypeptide is substantially similar to SEQ ID NO: 4, 6, 8, 10, 12, 14,16, or 18, wherein the polypeptide comprises an amino acid sequencehaving W or Y at position 28, and T at position 44, and wherein theencoded polypeptide is capable of binding or inhibiting activin A,myostatin or GDF-11.

Nucleic acid molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, synthetic DNA,DNA amplified by PCR, and combinations thereof. Genomic DNA may beisolated by conventional techniques, such as by using the DNA of SEQ IDNO: 3, 5, 11 or 13, or a suitable fragment thereof, as a probe. GenomicDNA encoding ActRIIB polypeptides is obtained from genomic librarieswhich are available for a number of species. Synthetic DNA is availablefrom chemical synthesis of overlapping oligonucleotide fragmentsfollowed by assembly of the fragments to reconstitute part or all of thecoding regions and flanking sequences. RNA may be obtained fromprocaryotic expression vectors which direct high-level synthesis ofmRNA, such as vectors using T7 promoters and RNA polymerase. cDNA isobtained from libraries prepared from mRNA isolated from various tissuesthat express ActRIIB. The DNA molecules of the invention include fulllength genes as well as polynucleotides and fragments thereof. The fulllength gene may also include sequences encoding the N-terminal signalsequence.

The invention further provides the nucleic acid molecule describe above,wherein the polynucleotide is operably linked to a transcriptional ortranslational regulatory sequence.

Exemplary polynucleotide and polypeptide sequences.svActRIIB (E28W, S44T) with signal sequence (SEQ ID NO: 3)atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtgagacacggtggtgcatctactacaacgccaactgggagctggagcgcaccaaccagaccggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgagggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagccccc accsvActRIIB (E28W, S44T) with signal sequence (SEQ ID NO: 4)mefglswvflvallrgvqcetrwciyynanwelertnqtglercegeqdkrlhcyaswrnssgtielvkkgcwlddfncydrqecvateenpqvyfcccegnfcnerfthlpeaggpevtyeppptaptsvActRIIB (E28W, S44T) without signal sequence (SEQ ID NO: 5)gagacacggtggtgcatctactacaacgccaactgggagctggagcgcaccaaccagaccggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgt gagggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccsvActRIIB (E28W, S44T) without signal sequence (SEQ ID NO: 6)etrwciyynanwelertnqtglercegeqdkrlhcyaswrnssgtielvkkgcwlddfncydrqecvateenpqvyfcccegnfcnerfthlpeag gpevtyeppptaptsvActRIIB-Fc (E28W, S44T) polynucleotide sequence with signal sequence(SEQ ID NO: 7) atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtgagacacggtggtgcatctactacaacgccaactgggagctggagcgcaccaaccagaccggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgagggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccggagggggaggatctgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctg aacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctcc ctgtctccgggtaaasvActRIIB-Fc (E28W, S44T) polypeptide sequence with signal sequence(SEQ ID NO: 8) mefglswvflvallrgvqcetrwciyynanwelertnqtglercegeqdkrlhcyaswrnssgtielvkkgcwlddfncydrqecvateenpqvyfcccegnfcnerfthlpeaggpevtyeppptaptggggsvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscs vmhealhnhytqkslslspgksvActRIIB-Fc (E28W, S44T) polynucleotidesequence without signal sequence (SEQ ID NO: 9)gagacacggtggtgcatctactacaacgccaactgggagctggagcgcaccaaccagaccggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgagggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccggagggggaggatctgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccg ggtaaasvActRIIB-Fc (E28W, S44T), polypeptide sequence  without signal sequence(SEQ ID NO: 10) etrwciyynanwelertnqtglercegeqdkrlhcyaswrnssgtielvkkgcwlddfncydrqecvateenpqvyfcccegnfcnerfthlpeaggpevtyeppptaptggggsvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsyltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslsp gksvActRIIB (E28Y, S44T) with signal sequence (SEQ ID NO: 11)atggagtttgggctgagctgggttttcctcgttgctatttaagaggtgtccagtgtgagacacggtactgcatctactacaacgccaactgggagctggagcgcaccaaccagaccggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgagggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagccccca ccsvActRIIB (E28Y, S44T) with signal sequence (SEQ ID NO: 12)mefglswvflvallrgvqcetryciyynanwelertnqtglercegeqdkrlhcyaswrnssgtielvkkgcwlddfncydrqecvateenpqvyfcccegnfcnerfthlpeaggpevtyeppptaptsvActRIIB (E28Y, S44T) without signal sequence (SEQ ID NO: 13)gagacacggtactgcatctactacaacgccaactgggagctggagcgcaccaaccagaccggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgagggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccsvActRIIB (E28Y, S44T) without signal sequence (SEQ ID NO: 14)etryciyynanwelertnqtglercegeqdkrlhcyaswrnssgtielvkkgcwlddfncydrqecvateenpqvyfcccegnfcnerfthlpeag gpevtyeppptaptsvActRIIB-Fc (E28Y, S44T) polynucleotide sequence with signal sequence(SEQ ID NO: 15) atggagtttgggctgagctgggttttcctcgttgctatttaagaggtgtccagtgtgagacacggtactgcatctactacaacgccaactgggagctggagcgcaccaaccagaccggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgagggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccggagggggaggatctgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccc tgtctccgggtaaasvActRIIB-Fc (E28Y, S44T) polypeptide sequence  with signal sequence(SEQ ID NO: 16) mefglswvflvallrgvqcetryciyynanwelertnqtglercegeqdkrlhcyaswrnssgtielvkkgcwlddfncydrqecvateenpqvyfcccegnfcnerfthlpeaggpevtyeppptaptggggsvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsyltvvhqdwingkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscs vmhealhnhytqkslslspgksvActRIIB-Fc (E28Y, S44T) polynucleotide sequence without signal sequence (SEQ ID NO: 17)gagacacggtactgcatctactacaacgccaactgggagctggagcgcaccaaccagaccggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgagggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccggagggggaggatctgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccg ggtaaasvActRIIB-Fc (E28Y, S44T) polypeptide sequence without signal sequence(SEQ ID NO: 18) etryciyynanwelertnqtglercegeqdkrlhcyaswrnssgtielvkkgcwlddfncydrqecvateenpqvyfcccegnfcnerfthlpeaggpevtyeppptaptggggsvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsyltvvhqdwingkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslsp gk 

In another aspect of the present invention, expression vectorscontaining the nucleic acid molecules and polynucleotides of the presentinvention are also provided, and host cells transformed with suchvectors, and methods of producing the svActRIIB polypeptides are alsoprovided. The term “expression vector” refers to a plasmid, phage, virusor vector for expressing a polypeptide from a polynucleotide sequence.Vectors for the expression of the svActRIIB polypeptides contain at aminimum sequences required for vector propagation and for expression ofthe cloned insert. An expression vector comprises a transcriptional unitcomprising an assembly of (1) a genetic element or elements having aregulatory role in gene expression, for example, promoters or enhancers,(2) a sequence that encodes svActRIIB polypeptides and proteins to betranscribed into mRNA and translated into protein, and (3) appropriatetranscription initiation and termination sequences. These sequences mayfurther include a selection marker. Vectors suitable for expression inhost cells are readily available and the nucleic acid molecules areinserted into the vectors using standard recombinant DNA techniques.Such vectors can include promoters which function in specific tissues,and viral vectors for the expression of svActRIIB polypeptides intargeted human or animal cells. An exemplary expression vector suitablefor expression of svActRIIB is the pDSRa, (described in WO 90/14363,herein incorporated by reference) and its derivatives, containingsvActRIIB polynucleotides, as well as any additional suitable vectorsknown in the art or described below.

The invention further provides methods of making svActRIIB polypeptides.A variety of other expression/host systems may be utilized. Thesesystems include but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid); or animal cell systems. Mammalian cells useful inrecombinant protein production include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, or theirderivatives such as Veggie CHO and related cell lines which grow inserum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31) orCHO strain DX-B11, which is deficient in DHFR (see Urlaub et al., 1980,Proc. Natl. Acad. Sci. USA 77:4216-20) COS cells such as the COS-7 lineof monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell23:175), W138, BHK, HepG2, 3T3 (ATCC CCL 163), RIN, MDCK, A549, PC12,K562, L cells, C127 cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNAcell line derived from the African green monkey kidney cell line CV1(ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), humanembryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermalA431 cells, human Colo205 cells, other transformed primate cell lines,normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, primary explants, HL-60, U937, HaK or Jurkat cells.Mammalian expression allows for the production of secreted or solublepolypeptides which may be recovered from the growth medium.

Using an appropriate host-vector system, svActRIIB polypeptides areproduced recombinantly by culturing a host cell transformed with anexpression vector containing the nucleic acid molecules of the presentinvention under conditions allowing for production. Transformed cellscan be used for long-term, high-yield polypeptide production. Once suchcells are transformed with vectors that contain selectable markers aswell as the desired expression cassette, the cells can be allowed togrow in an enriched media before they are switched to selective media,for example. The selectable marker is designed to allow growth andrecovery of cells that successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell line employed. Anoverview of expression of recombinant proteins is found in Methods ofEnzymology, v. 185, Goeddell, D. V., ed., Academic Press (1990).

In some cases, such as in expression using procaryotic systems, theexpressed polypeptides of this invention may need to be “refolded” andoxidized into a proper tertiary structure and disulfide linkagesgenerated in order to be biologically active. Refolding can beaccomplished using a number of procedures well known in the art. Suchmethods include, for example, exposing the solubilized polypeptide to apH usually above 7 in the presence of a chaotropic agent. The selectionof chaotrope is similar to the choices used for inclusion bodysolubilization, however a chaotrope is typically used at a lowerconcentration. Exemplary chaotropic agents are guanidine and urea. Inmost cases, the refolding/oxidation solution will also contain areducing agent plus its oxidized form in a specific ratio to generate aparticular redox potential which allows for disulfide shuffling to occurfor the formation of cysteine bridges. Some commonly used redox couplesinclude cysteine/cystamine, glutathione/dithiobisGSH, cupric chloride,dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME.In many instances, a co-solvent may be used to increase the efficiencyof the refolding. Commonly used cosolvents include glycerol,polyethylene glycol of various molecular weights, and arginine.

In addition, the polypeptides can be synthesized in solution or on asolid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. See, for example, Stewart and Young,Solid Phase Peptide Synthesis, 2d. Ed., Pierce Chemical Co. (1984); Tamet al., J Am Chem Soc, 105:6442, (1983); Merrifield, Science 232:341-347(1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds,Academic Press, New York, 1-284; Barany et al., Int J Pep Protein Res,30:705-739 (1987).

The polypeptides and proteins of the present invention can be purifiedaccording to protein purification techniques are well known to those ofskill in the art. These techniques involve, at one level, the crudefractionation of the proteinaceous and non-proteinaceous fractions.Having separated the peptide polypeptides from other proteins, thepeptide or polypeptide of interest can be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). The term“isolated polypeptide” or “purified polypeptide” as used herein, isintended to refer to a composition, isolatable from other components,wherein the polypeptide is purified to any degree relative to itsnaturally-obtainable state. A purified polypeptide therefore also refersto a polypeptide that is free from the environment in which it maynaturally occur. Generally, “purified” will refer to a polypeptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a peptide or polypeptidecomposition in which the polypeptide or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 85%, or about 90% or more of the proteins inthe composition.

Various techniques suitable for use in purification will be well knownto those of skill in the art. These include, for example, precipitationwith ammonium sulphate, PEG, antibodies (immunoprecipitation) and thelike or by heat denaturation, followed by centrifugation; chromatographysuch as affinity chromatography (Protein-A columns), ion exchange, gelfiltration, reverse phase, hydroxylapatite, hydrophobic interactionchromatography, isoelectric focusing, gel electrophoresis, andcombinations of these techniques. As is generally known in the art, itis believed that the order of conducting the various purification stepsmay be changed, or that certain steps may be omitted, and still resultin a suitable method for the preparation of a substantially purifiedpolypeptide. Exemplary purification steps are provided in the Examplesbelow.

Various methods for quantifying the degree of purification ofpolypeptide will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificbinding activity of an active fraction, or assessing the amount ofpeptide or polypeptide within a fraction by SDS/PAGE analysis. Apreferred method for assessing the purity of a polypeptide fraction isto calculate the binding activity of the fraction, to compare it to thebinding activity of the initial extract, and to thus calculate thedegree of purification, herein assessed by a “-fold purificationnumber.” The actual units used to represent the amount of bindingactivity will, of course, be dependent upon the particular assaytechnique chosen to follow the purification and whether or not thepolypeptide or peptide exhibits a detectable binding activity.

Stabilized activin type IIB polypeptides bind to ligands that activatemuscle-degradation cascades. svActRIIB polypeptides capable of bindingand inhibiting the activity of the ligands activin A, myostatin, and/orGDF-11, and have the ability to treat diseases that involve muscleatrophy, as well as the treatment of certain cancers, and otherdiseases.

The Examples below show improved properties for svActRIIB polypeptidesand proteins having the amino acid substitutions described herein, whileretaining the ability to bind and neutralize myostatin, activin A, orGDF-11 in in vitro assays, as well as retaining in vivo activity. Theseproperties result in proteins and polypeptides having improvedmanufacturability in comparison to other soluble receptors.

Antibodies

The present invention further includes antibodies which bind tostabilized ActRIIB polypeptides, including those that specifically bindto the svActRIIB polypeptides of the present invention. As used hereinthe term “specifically binds” refers to antibodies having a bindingaffinity (K_(a)) for svActRIIB polypeptides of 10⁶ M⁻¹ or greater. Asused herein, the term “antibody” refers to intact antibodies includingpolyclonal antibodies (see, for example Antibodies: A Laboratory Manual,Harlow and Lane (eds), Cold Spring Harbor Press, (1988)), and monoclonalantibodies (see, for example, U.S. Pat. Nos. RE 32,011, 4,902,614,4,543,439, and 4,411,993, and Monoclonal Antibodies: A New Dimension inBiological Analysis, Plenum Press, Kennett, McKearn and Bechtol (eds.)(1980)). As used herein, the term “antibody” also refers to a fragmentof an antibody such as F(ab), F(ab′), F(ab′)₂, Fv, Fc, and single chainantibodies which are produced by recombinant DNA techniques or byenzymatic or chemical cleavage of intact antibodies. The term “antibody”also refers to bispecific or bifunctional antibodies, which are anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites. Bispecific antibodies can be producedby a variety of methods including fusion of hybridomas or linking ofFab′ fragments. (See Songsivilai et al, Clin. Exp. Immunol. 79:315-321(1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992)).

As used herein the term “antibody” also refers to chimeric antibodies,that is, antibodies having a human constant antibody immunoglobin domaincoupled to one or more non-human variable antibody immunoglobin domain,or fragments thereof (see, for example, U.S. Pat. Nos. 5,595,898 and5,693,493). Antibodies also refers to “humanized” antibodies (see, forexample, U.S. Pat. No. 4,816,567 and WO 94/10332), minibodies (WO94/09817), maxibodies, and antibodies produced by transgenic animals, inwhich a transgenic animal containing a proportion of the human antibodyproducing genes but deficient in the production of endogenous antibodiesare capable of producing human antibodies (see, for example, Mendez etal., Nature Genetics 15:146-156 (1997), and U.S. Pat. No. 6,300,129).The term “antibodies” also includes multimeric antibodies, or a higherorder complex of proteins such as heterdimeric antibodies, andanti-idiotypic antibodies. “Antibodies” also includes anti-idiotypicantibodies. The antibodies against sv ActRIIB polypeptides can be used,for example, to identify and quantitate svActRIIB in vitro and in vivo.

Also included are polyclonal antibodies from any mammal, for examplemouse and rat antibodies, and rabbit antibodies, that bind specificallyto the svActRIIB polypeptides described herein.

Such antibodies find use as research tools and in quantitative assaysfor detecting and assaying the polypeptides disclosed herein. Suchantibodies are made using methods described above and as known in theart.

Pharmaceutical Compositions

Pharmaceutical compositions containing the svActRIIB proteins andpolypeptides of the present invention are also provided. Suchcompositions comprise a therapeutically or prophylactically effectiveamount of the polypeptide or protein in admixture with pharmaceuticallyacceptable materials, and physiologically acceptable formulationmaterials. The pharmaceutical composition may contain formulationmaterials for modifying, maintaining or preserving, for example, the pH,osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption or penetration ofthe composition. Suitable formulation materials include, but are notlimited to, amino acids (such as glycine, glutamine, asparagine,arginine or lysine); antimicrobials; antioxidants (such as ascorbicacid, sodium sulfite or sodium hydrogen-sulfite); buffers (such asborate, bicarbonate, Tris-HCl, citrates, phosphates, other organicacids); bulking agents (such as mannitol or glycine), chelating agents(such as ethylenediamine tetraacetic acid (EDTA)); complexing agents(such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides and other carbohydrates (such as glucose, mannose, ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring; flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides(preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18^(th) Edition, A. R. Gennaro,ed., Mack Publishing Company, 1990).

The optimal pharmaceutical composition will be determined by one skilledin the art depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See for example,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the polypeptide. For example, suitablecompositions may be water for injection, physiological saline solutionfor parenteral administration.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffers, or acetate buffers, which may further include sorbitol or asuitable substitute thereof. In one embodiment of the present invention,compositions may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (Remington's Pharmaceutical Sciences, supra) in theform of a lyophilized cake or an aqueous solution. Further, thetherapeutic composition may be formulated as a lyophilizate usingappropriate excipients such as sucrose.

The formulations can be delivered in a variety of methods, for example,by inhalation therapy, orally, or by injection. When parenteraladministration is contemplated, the therapeutic compositions for use inthis invention may be in the form of a pyrogen-free, parenterallyacceptable aqueous solution comprising the desired polypeptide in apharmaceutically acceptable vehicle. A particularly suitable vehicle forparenteral injection is sterile distilled water in which a polypeptideis formulated as a sterile, isotonic solution, properly preserved. Yetanother preparation can involve the formulation of the desired moleculewith an agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (polylactic acid, polyglycolic acid), beads, orliposomes, that provides for the controlled or sustained release of theproduct which may then be delivered via a depot injection. Hyaluronicacid may also be used, and this may have the effect of promotingsustained duration in the circulation. Other suitable means for theintroduction of the desired molecule include implantable drug deliverydevices.

In another aspect, pharmaceutical formulations suitable for injectableadministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions. In anotherembodiment, a pharmaceutical composition may be formulated forinhalation. Inhalation solutions may also be formulated with apropellant for aerosol delivery. In yet another embodiment, solutionsmay be nebulized. Pulmonary administration is further described in PCTApplication No. PCT/US94/001875, which describes pulmonary delivery ofchemically modified proteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, molecules that areadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the therapeutic molecule. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed. Pharmaceutical compositions for oral administration can alsobe formulated using pharmaceutically acceptable carriers well known inthe art in dosages suitable for oral administration. Such carriersenable the pharmaceutical compositions to be formulated as tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions,and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations that can be used orally also includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving polypeptides in sustained-or controlled-delivery formulations. Techniques for formulating avariety of other sustained- or controlled-delivery means, such asliposome carriers, bio-erodible microparticles or porous beads and depotinjections, are also known to those skilled in the art. See for example,PCT/US93/00829 that describes controlled release of porous polymericmicroparticles for the delivery of pharmaceutical compositions.Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15:167-277, (1981); Langer et al., Chem. Tech., 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomes, which can be prepared by any ofseveral methods known in the art. See e.g., Eppstein et al., PNAS (USA),82:3688 (1985); EP 36,676; EP 88,046; EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which thepolypeptide is being used, the route of administration, and the size(body weight, body surface or organ size) and condition (the age andgeneral health) of the patient. Accordingly, the clinician may titer thedosage and modify the route of administration to obtain the optimaltherapeutic effect. A typical dosage may range from about 0.1 mg/kg toup to about 100 mg/kg or more, depending on the factors mentioned above.Polypeptide compositions may be preferably injected or administeredintravenously. Long-acting pharmaceutical compositions may beadministered every three to four days, every week, or biweekly dependingon the half-life and clearance rate of the particular formulation. Thefrequency of dosing will depend upon the pharmacokinetic parameters ofthe polypeptide in the formulation used. Typically, a composition isadministered until a dosage is reached that achieves the desired effect.The composition may therefore be administered as a single dose, or asmultiple doses (at the same or different concentrations/dosages) overtime, or as a continuous infusion. Further refinement of the appropriatedosage is routinely made. Appropriate dosages may be ascertained throughuse of appropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional routes, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, or intraperitoneal; as wellas intranasal, enteral, topical, sublingual, urethral, vaginal, orrectal means, by sustained release systems or by implantation devices.Where desired, the compositions may be administered by bolus injectionor continuously by infusion, or by implantation device. Alternatively oradditionally, the composition may be administered locally viaimplantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Wherean implantation device is used, the device may be implanted into anysuitable tissue or organ, and delivery of the desired molecule may bevia diffusion, timed-release bolus, or continuous administration.

In some cases, the svActRIIB polypeptides of the present invention canbe delivered by implanting certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptide. Such cells may be animal or human cells, andmay be autologous, heterologous, or xenogeneic. Optionally, the cellsmay be immortalized. In order to decrease the chance of an immunologicalresponse, the cells may be encapsulated to avoid infiltration ofsurrounding tissues. The encapsulation materials are typicallybiocompatible, semi-permeable polymeric enclosures or membranes thatallow the release of the polypeptide product(s) but prevent thedestruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

svActRIIB gene therapy in vivo is also envisioned wherein a nucleic acidmolecule encoding svActRIIB, or a derivative of svActRIIB is introduceddirectly into the subject. For example, a nucleic acid sequence encodinga svActRIIB is introduced into target cells via local injection of anucleic acid construct with or without an appropriate delivery vector,such as an adeno-associated virus vector. Alternative viral vectorsinclude, but are not limited to, retroviruses, adenovirus, herpessimplex, virus and papilloma virus vectors. Physical transfer of thevirus vector may be achieved in vivo by local injection of the desirednucleic acid construct or other appropriate delivery vector containingthe desired nucleic acid sequence, liposome-mediated transfer, directinjection (naked DNA), or microparticle bombardment (gene-gun).

The compositions of the present disclosure may be used alone or incombination with other therapeutic agents to enhance their therapeuticeffects or decrease potential side effects.

Uses of svActRIIB Compositions

The present invention provides methods and pharmaceutical compositionsfor reducing or neutralizing the amount or activity of myostatin,activin A, or GDF-11 in vivo and in vitro. svActRIIB polypeptides have ahigh binding affinity for myostatin, activin A, and GDF-11, and arecapable of reducing and inhibiting the biological activities of at leastone of myostatin, activin A and GDF-11.

In one aspect, the present invention provides methods and reagents fortreating myostatin-related and/or activin A related disorders in asubject in need of such a treatment by administering an effective dosageof an svActRIIB composition to the subject. As used herein the term“subject” refers to any animal, such as mammals including humans.

The compositions of the present invention are useful for increasing leanmuscle mass in a subject. The compositions may also be useful toincrease lean muscle mass in proportion to fat mass, and thus decreasefat mass as percentage of body weight in a subject. Example 3demonstrates that the svActRIIB polypeptides and proteins of theinvention can increase lean muscle mass in animals.

The disorders that can be treated by an svActRIIB composition includebut are not limited to various forms of muscle wasting, as well asmetabolic disorders such as diabetes and related disorders, and bonedegenerative diseases such as osteoporosis.

Muscle wasting disorders also include dystrophies such as Duchenne'smuscular dystrophy, progressive muscular dystrophy, Becker's typemuscular dystrophy, Dejerine-Landouzy muscular dystrophy, Erb's musculardystrophy, and infantile neuroaxonal muscular dystrophy. Additionalmuscle wasting disorders arise from chronic diseases or disorders suchas amyotrophic lateral sclerosis, congestive obstructive pulmonarydisease, cancer, AIDS, renal failure, organ atrophy, androgendeprivation, and rheumatoid arthritis.

Over-expression of myostatin and/or activin may contribute to cachexia,a severe muscle wasting syndrome. Cachexia results from cancers, andalso arises due to rheumatoid arthritis, diabetic nephropathy, renalfailure, chemotherapy, injury due to burns, as well as other causes. Inanother example, serum and intramuscular concentrations ofmyostatin-immunoreactive protein was found to be increased in menexhibiting AIDS-related muscle wasting and was inversely related tofat-free mass (Gonzalez-Cadavid et al., PNAS USA 95: 14938-14943(1998)). Myostatin levels have also been shown to increase in responseto burns injuries, resulting in a catabolic muscle effect (Lang et al,FASEB J 15, 1807-1809 (2001)). Additional conditions resulting in musclewasting may arise from inactivity due to disability such as confinementin a wheelchair, prolonged bed rest due to stroke, illness, spinal chordinjury, bone fracture or trauma, and muscular atrophy in a microgravityenvironment (space flight). For example, plasma myostatin immunoreactiveprotein was found to increase after prolonged bed rest (Zachwieja et al.J Gravit Physiol. 6(2):11(1999). It was also found that the muscles ofrats exposed to a microgravity environment during a space shuttle flightexpressed an increased amount of myostatin compared with the muscles ofrats which were not exposed (Lalani et al., J. Endocrin 167 (3):417-28(2000)).

In addition, age-related increases in fat to muscle ratios, andage-related muscular atrophy appear to be related to myostatin. Forexample, the average serum myostatin-immunoreactive protein increasedwith age in groups of young (19-35 yr. old), middle-aged (36-75 yr.old), and elderly (76-92 yr old) men and women, while the average musclemass and fat-free mass declined with age in these groups (Yarasheski etal. J Nutr Aging 6(5):343-8 (2002)). In addition, myostatin has now beenfound to be expressed at low levels in heart muscle and expression isupregulated in cardiomyocytes after infarct (Sharma et al., J CellPhysiol. 180 (1):1-9 (1999)). Therefore, reducing myostatin levels inthe heart muscle may improve recovery of heart muscle after infarct.

Myostatin also appears to influence metabolic disorders including type 2diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, andobesity. For example, lack of myostatin has been shown to improve theobese and diabetic phenotypes of two mouse models (Yen et al. FASEB J.8:479 (1994). The svActRIIB polypeptides of the present disclosure aresuitable for treating such metabolic disorders. Therefore, administeringthe compositions of the present invention will improve diabetes,obesity, and hyperglycemic conditions in suitable subjects. In addition,compositions containing the svActRIIB polypeptides may decrease foodintake in obese individuals.

Administering the stabilized ActRIIB polypeptides of the presentinvention may improve bone strength and reduce osteoporosis and otherdegenerative bone diseases. It has been found, for example, thatmyostatin-deficient mice showed increased mineral content and density ofthe mouse humerus and increased mineral content of both trabecular andcortical bone at the regions where the muscles attach, as well asincreased muscle mass (Hamrick et al. Calcif Tissue Int 71(1):63-8(2002)). In addition, the svActRIIB compositions of the presentinvention can be used to treat the effects of androgen deprivation incases such as androgen deprivation therapy used for the treatment ofprostate cancer, for example.

The present invention also provides methods and compositions forincreasing muscle mass in food animals by administering an effectivedosage of the svActRIIB proteins to the animal. Since the matureC-terminal myostatin polypeptide is similar or identical in all speciestested, svActRIIB polypeptides would be expected to be effective forincreasing lean muscle mass and reducing fat in any agriculturallyimportant species including cattle, chicken, turkeys, and pigs.

The svActRIIB polypeptides and compositions of the present inventionalso antagonize the activity of activin A, as shown in the in vitroassays below. Activin A is known to be expressed in certain types ofcancers, particularly gonadal tumors such as ovarian carcinomas, and tocause severe cachexia. (Ciprano et al. Endocrinol 141 (7):2319-27(2000), Shou et al., Endocrinol 138 (11):5000-5 (1997); Coerver et al,Mol Endocrinol 10(5):534-43 (1996); Ito et al. British J Cancer82(8):1415-20 (2000), Lambert-Messerlian, et al, Gynecologic Oncology74:93-7 (1999). Therefore, the compositions of the present disclosuremay be used to treat conditions related to activin A overexpression, aswell as myostatin expression, such as cachexia from certain cancers andthe treatment of certain gonadal type tumors.

In addition, the svActRIIB polypeptides of the present invention areuseful for detecting and quantitating myostatin, activin A, or GDF-11 inany number of assays. In general, the stabilized ActRIIB polypeptides ofthe present invention are useful as capture agents to bind andimmobilize myostatin, activin A, or GDF-11 in a variety of assays,similar to those described, for example, in Asai, ed., Methods in CellBiology, 37, Antibodies in Cell Biology, Academic Press, Inc., New York(1993). The polypeptides may be labeled in some manner or may react witha third molecule such as an antibody which is labeled to enablemyostatin to be detected and quantitated. For example, a polypeptide ora third molecule can be modified with a detectable moiety, such asbiotin, which can then be bound by a fourth molecule, such asenzyme-labeled streptavidin, or other proteins. (Akerstrom, J Immunol135:2589 (1985); Chaubert, Mod Pathol 10:585 (1997)).

The invention having been described, the following examples are offeredby way of illustration, and not limitation.

Example 1 Expression and Purification of svActRIIB Polypeptides

The following methods were used for expressing and purifying thestabilized ActRIIB polypeptides.

The cDNA of the human activin type IIB receptor was isolated from a cDNAlibrary of human testis origin (Clontech, Inc.) and cloned as describedin U.S. application Ser. No. 11/590,962, U.S. application publicationNo: 2007/0117130, which is herein incorporated by reference.

The following method was used to produce the svActRIIB-Fc (E28W, S44T)polypeptide (SEQ ID NO: 10), and the ActRIIB-Fc (E28W) (SEQ ID NO: 21).Polynucleotides encoding the svActRIIB, (E28W, S44T) (SEQ ID NO: 5), orpolynucleotides encoding ActRIIB (E28W) (SEQ ID NO: 19) were fused topolynucleotides encoding the human IgG2 Fc (SEQ ID NO: 22), viapolynucleotides encoding hinge linker sequence (SEQ ID NO: 26) using PCRoverlap extension using primers containing the mutation resulting in theamino acid substitutions at position 28 of E to W, and at position 44 ofS to T. The full polynucleotide sequence is SEQ ID NO: 9 forsvActRIIB-IgG Fc (E28W, S44T), and SEQ ID NO: 20 for ActRIIB-ActRIIB-IgGFc (E28W). Double stranded DNA fragments were subcloned into vectorspTT5 (Biotechnology Research Institute, National Research Council Canada(NRCC), 6100 Avenue Royalmount, Montreal (Québec) Canada H4P 2R2),pDSRα□□ described in WO/9014363) and/or derivatives of pDSRα.

Transient expression of stabilized ActRIIB-Fc polypeptides was carriedout as follows.

The svActRIIB-IgG Fc (E28W, S44T) (SEQ ID NO: 10), and ActRIIB-IgG Fc(E28W) (SEQ ID NO: 21) polypeptides were expressed transiently inserum-free suspension adapted 293-6E cells (National Research Council ofCanada, Ottawa, Canada) maintained in FreeStyle™ medium (InvitrogenCorporation, Carlsbad, Calif.) supplemented with 250 μg/ml geneticin(Invitrogen) and 0.1% Pluronic F68 (Invitrogen). Transfections wereperformed as 1 L cultures. Briefly, the cell inoculum was grown to1.1×10⁶ cells/ml in a 4 L fernbach shake flask (Corning, Inc.). Theshake flask culture was maintained on an Innova 2150 shaker platform(News Brunswick Scientific, Edison, N.J.) at 65 RPM which was placed ina humidified incubator maintained at 37° C. and 5% CO₂. At the time oftransfection, the 293-6E cells were diluted to 1.0×10⁶ cells/ml.

The transfection complexes were formed in 100 ml FreeStyle™ 293 Media(Invitrogen). 1 mg plasmid DNA was first added to the medium followed by3 ml of FuGene HD transfection reagent (Roche Applied Science,Indianapolis, Ind.). The transfection complex was incubated at roomtemperature for approximately 15 minutes and then added to the cells inthe shake flask. Twenty hours post transfection, 20% (w/v) of peptoneTN1 (OrganoTechnie S. A., TeknieScience, QC, Canada) was added to reacha final concentration of 0.5% (w/v). The transfection/expression wasperformed for 4-7 days, after which the conditioned medium was harvestedby centrifugation at 4,000 RPM for 60 minutes at 4° C.

Stable transfection and expression was carried out as follows. ThesvActRIIB-IgG-Fc cell lines were created by transfecting stable CHO hostcells with the expression plasmids containing polynucleotides encodingsvActRIIB-IgG Fc (E28W, S44T) (SEQ ID NO: 9) or ActRIIB-IgG Fc (E28W)(SEQ ID NO: 20) using a standard electroporation procedure. Aftertransfection of the host cell line with the expression plasmids thecells were grown in serum-free selection medium without GHT for 2-3weeks to allow for selection of the plasmid and recovery of the cells.Cells are selected until they achieved greater than 85% viability. Thispool of transfected cells was then cultured in medium containing 150 nMmethotrexate.

In a six-day expression assay, pools of svActRIIB-Fc (E28W, S44T)expressing cells showed higher cell titer, growth performance, andimproved specific productivity (picogram/cell/day) of protein producedcompared with pools of ActRIIB-Fc (E28W) expressing cells. Select pools,for example, produced about 1.2 g/liter for svActRIIB-Fc (E28W, S44T)compared with 0.9 g/liter for ActRIIB-Fc (E28W).

Each of an svActRIIB-Fc (E28W, S44T) and an ActRIIB-Fc (E28W) expressingcell line was scaled up using a typical fed-batch process. Cells wereinoculated into a Wave bioreactor (Wave Biotech LLC). Cultures were fedthree times with bolus feeds. 10 L were harvested on day 10, theremainder was harvested on day 11; both harvests underwent depthfiltration followed by sterile filtration. The conditioned media wasfiltered through a 10 inch 0.45/0.2 micron pre filter, followed by afiltration through a 6 inch 0.2 micron filter.

Protein Purification

Approximately 5 L of conditioned media was directly loaded onto a 220 mLMabSelect™ column Protein A column (GE Healthcare). The column waspre-equilibrated in PBS (phosphate-buffered saline: 2.67 mM potassiumchloride, 138 mM sodium chloride, 1.47 mM potassium phosphate monobasic,8.1 mM sodium phosphate dibasic, pH 7.4). The column was washed with theequilibration buffer until the reading at OD280 was approximately zero,and then the protein was eluted with 0.1M acetic acid.

The Mabselect™ Pool was applied to a 300 mL SP-HP column (GE Healthcare)(5×15 cm). The column was pre-equilibrated with 10 mM NaOAC, pH 5. Thecolumn was then washed with the equilibration buffer until the readingat OD280 was approximately 0. The column was eluted with 20 columnvolumes of a gradient buffer from 0-150 mM NaCl in 10 mM NaOAC, pH 5.The SP-HP pool was concentrated, and filtered with a 0.2 uM celluloseacetate (Corning) filter.

The sequences of the proteins used are set forth in the Table below.

ActRIIB- ActRIIB Linker- Fc sequence Hinge IgG2 Fc svActRIIB-ETRWCIYYNANWELE GGGGSV APPVAGPSVFLFPPK IgG₂Fc RTNQTGLERCEGEQD ECPPCPPKDTLMISRTPEVTC (E28W, KRLHCYASWRNSSGT (SEQ ID VVVDVSHEDPEVQFN S44T)IELVKKGCWLDDFNC NO: 27) WYVDGVEVHNAKTKP (SEQ ID YDRQECVATEENPQVREEQFNSTFRVVSVL NO: 10) YFCCCEGNFCNERFT TVVHQDWLNGKEYKC HLPEAGGPEVTYEPP KVSNKGLPAPIEKTI PTAPT SKTKGQPREPQVYTL (SEQ ID NO: 6) PPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPP MLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 22) ActRIIB- ETRWCIYYNANWELE GGGGSVAPPVAGPSVFLFPPK IgG₂Fc RTNQSGLERCEGEQD ECPPCP PKDTLMISRTPEVTC (E28W)KRLHCYASWRNSSGT (SEQ ID VVVDVSHEDPEVQFN (SEQ ID IELVKKGCWLDDFNC NO: 27)WYVDGVEVHNAKTKP NO: 21) YDRQECVATEENPQV REEQFNSTFRVVSVL YFCCCEGNFCNERFTTVVHQDWLNGKEYKC HLPEAGGPEVTYEPP KVSNKGLPAPIEKTI PTAPT SKTKGQPREPQVYTL(SEQ ID NO: 19) PPSREEMTKNQVSLT CLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCS VMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 22)

Example 2 Characterization of Polypeptides

Samples of the svActRIIB-Fc (E28W, S44T) (SEQ ID NO: 10) purifiedthrough the MabSelect™ step, and ActRIIB-Fc (E28W) (SEQ ID NO: 21)polypeptides purified through the SP-HP column step, as described above,were diluted with PBS, pH 7.4 to 0.2 mg/ml. The glycosylation profile ofthe polypeptides were then determined using SEC as described below.

Size Exclusion Chromatography (SEC).

Experiments were performed on an Agilent 1100 HPLC system with twocolumns (TOSOHAAS G3000swxl, 7.8×300 mm) in tandem. 2× PBS was used asthe mobile phase at 0.5 ml/minute.

FIG. 1 shows a comparison between ActRIIB-Fc (E28W) and svActRIIB-Fc(E28W, S44T) on an SEC column using the protocols described above.svActRIIB-Fc (E28W, S44T) shows a single peak compared with ActRIIB-Fc(E28W), which shows three peaks. These correspond to the degree ofN-linked glycosylation at the N42 position of the Fc dimers of bothproteins. The single peak of the svActRIIB-Fc (E28W, S44T) polypeptidecorresponds to fully glycosylated N-linked asparagines at position N42of the dimer. The three peaks of ActRIIB-Fc (E28W) corresponds to (fromleft to right), fully glycosylated asparagines at N42, partiallyglycosylated asparagines at N42, and non-glycosylated asparagines atN42. Therefore, this demonstrates that the svActRIIB-Fc (E28W, S44T)molecule is fully glycosylated compared to ActRIIB-Fc (E28W), which isheterogeneous with respect to this glycosylation site, and thus moredifficult to purify. In addition, preliminary studies indicate that thesvActRIIB-Fc (E28W, S44T) molecule has addition improvedmanufacturability properties as set forth below. Additional studies alsodemonstrated that the least glycosylated peak of the ActRIIB-Fc (E28W)has lower physical and thermal stability than partially and fullyglycosylated molecules.

Determination of K_(D) and IC₅₀ values of the receptor polypeptides foractivin A, myostatin, and GDF-11 were obtained as described below.

KinEx A™ Equilibrium Assays

Solution-based equilibrium-binding assays using KinExA™ technology(Sapidyne Instruments, Inc.) were used to determine the dissociationequilibrium (K_(D)) of ligand binding to ActRIIB-Fc polypeptides.UltraLink Biosupport beads (Pierce) was pre-coated with about 100 μg/mleach of myostatin, GDF-11, and activin A overnight, and then blockedwith BSA. 1 pM and 3 pM of ActRIIB-Fc (E28W) (SEQ ID NO: 21) andsvActRIIB-Fc (E28W, S44T) (SEQ ID NO: 10) samples were incubated withvarious concentrations (0.7 fM to 160 pM) of myostatin, activin A, andGDF-11 respectively in sample buffer at room temperature for 8 hoursbefore being run through the ligand-coated beads. The amount of thebead-bound soluble receptor was quantified by fluorescent (Cy5) labeledgoat anti-human-Fc antibody at 1 mg/ml in superblock. The binding signalis proportional to the concentration of free soluble receptor atequilibrium with a given myostatin, activin A, or GDF-11 concentration.K_(D) was obtained from the nonlinear regression of the competitioncurves using a dual-curve one-site homogeneous binding model provided inthe KinEx A™ software (Sapidyne Instruments, Inc.). The K_(D) valuesobtained for each are given in the table below.

Myostatin GDF-11 Activin A ActRIIB-Fc (E28W) 0.1 pM 0.1 pM 0.2 pMsvActRIIB-Fc 0.1 pM 0.1 pM 0.1 pM (E28W, S44T)

C2C12 Cell Based Activity Assay

The ability of ActRIIB-Fc (E28W) (SEQ ID NO: 21) and svActRIIB-Fc (E28W,S44T) (SEQ ID NO: 10) to inhibit the binding of activin A, GDF-11, ormyostatin to the wild type activin IIB receptor-Fc was tested using acell based activity assay as described below.

A myostatin/activin/GDF-11-responsive reporter cell line was generatedby transfection of C2C12 myoblast cells (ATCC No: CRL-1772) with apMARE-luc construct. The pMARE-luc construct is made by cloning twelverepeats of the CAGA sequence, representing the myostatin/activinresponse elements (Dennler et al. EMBO 17: 3091-3100 (1998)) into apLuc-MCS reporter vector (Stratagene cat #219087) upstream of the TATAbox. The C2C12 cells naturally express activin receptor IIB on theircell surface. When myostatin/activinA/GDF-11 binds the cell receptors,the Smad pathway is activated, and phosphorylated Smad binds to theresponse element (Macias-Silva et al. Cell 87:1215 (1996)), resulting inthe expression of the luciferase gene. Luciferase activity was thenmeasured using a commercial luciferase reporter assay kit (cat # E4550,Promega, Madison, Wis.) according to manufacturer's protocol. A stableline of C2C12 cells that has been transfected with pMARE-luc(C2C12/pMARE) was used to measure activity according to the followingprocedure. Reporter cells were plated into 96 well cultures. Screeningusing dilutions of the ActRIIB-IgG2 Fc fusions constructed as describedabove was performed with the concentration fixed at 4 nM activin A,myostatin, and GDF-11. Each of these ligands was pre-incubated with thereceptors at several concentrations. Activity was measured bydetermining the luciferase activity in the treated cultures. The IC₅₀values were determined for each polypeptide. These are shown in theTable below. These values are given in Table below.

Myostatin GDF-11 Activin A ActRIIB-Fc (E28W) 0.95 nM 2.4 nM 3.2 nMsvActRIIB-Fc 1.07 nM 2.4 nM 3.6 nM (E28W, S44T)

Thus the cell based activities are approximately the same for ActRIIB-Fc(E28W) and svActRIIB-Fc (E28W, S44T).

Stability at Low pH

Stability of a protein at low pH is a useful parameter in consideringthe manufacturability of the protein, since the viral inactivation stepof a commercial production process typically is carried out at low pH,such as between about pH 3.0 to 4.0.

To assess the short term protein stability effects at low pH experiencedduring the viral inactivation step of commercial protein purificationthe following test was performed. Each protein was diluted to 10 mg/mlof 100 mM sodium acetate, pH 3.5. This was stored at 25° C. and analyzedat time 0, at 2 hours and at 24 hours using SEC analysis. SEC analysiswas performed as described above, and percentage of high molecularweight aggregates was determined.

% HMW aggregate T = 0 T = 2 hours T = 4 hours ActRIIB-Fc (E28W) 1.531.36 13.74 svActRIIB-Fc 1.66 2.17 8.93 (E28W, S44T)

Thus the percentage of high molecular weight aggregates produced at pH3.5 is substantially less for svActRIIB-Fc (E28W, S44T) than ActRIIB-Fc(E28W) at 4 hours.

Additional studies showed that svActRIIB-Fc (E28W, S44T) showed betterreversibility than ActRIIB-Fc (E28W) from exposure to pH 3.0, 3.5 and5.0, and that svActRIIB-Fc (E28, S44T) was more homogeneous thatActRIIB-Fc (E28W) at all pHs.

Thus, the svActRIIB-Fc (E28W, S44T) polypeptides are demonstrated tohave improved manufacturability characteristics, in particular, improvedstability at low pH, and greater homogeneity at all pHs compared withActRIIB-Fc (E28W) while retaining the ability to inhibit activin A,myostatin, and GDF-11 activity.

Example 3 Determination of In Vivo Efficacy

11-week-old female C57B1/6 mice were purchased from Charles RiverLaboratories. The mice (ten mice per group) were administered a singledose (10 mg/kg) of svActRIIB-Fc (E28W, S44T) (SEQ ID NO: 10) or vehicle(PBS). Lean body mass was determined by NMR (PIXImus, GE LUNARCorporation) at 3, 7, 10 and 14 days after dose administration for theten animals in each group. The results for each set of mice are shown inFIG. 2. It can be seen that a single dose of svActRIIB-Fc (E28W, S44T)significantly increased lean body mass in the animals. (P<0.001, basedon repeated measurement ANOVA. n=10 animals per group).

A study to determine dose-response efficacy was carried out as follows.Escalating single doses of 0, 0.3, 3, 10, and 30 mg/kg of svActRIIB-Fc(E28W, S44T) (SEQ ID NO: 10) in PBS was administered subcutaneously tofemale 10-12 week old C57B1/6 mice (Charles River Laboratories). Sixanimals were initially in each dosage group including the PBS controlgroup. Lean body mass was determined by NMR (PIXImus, GE LUNARCorporation) every two to four days for the forty-two days of the study.At the end of each week, one animal from each group was sacrificed toobtain additional data (six in total each week from all six groups), andthe lean body mass determined for the remaining animals in subsequentweeks. The results are set out in FIG. 3. It can be seen that thesvActRIIB-Fc (E28W, S44T) polypeptide at all doses significantlyincreased muscle mass in the animals, in a dose-dependent manner.

In further studies, head to head comparisons between ActRIIB-Fc (E28W)(SEQ ID NO: 21) and svActRIIB-Fc (E28W, S44T) (SEQ ID NO: 10) wereperformed on female C57B1/6 mice (Charles River Laboratories, 10 animalsper group) to measure the increase in lean muscle mass and body weightchanges after a single dose of 10 mg/kg of each soluble receptorcompared with a control group (administered PBS). Lean body mass wasdetermined by NMR (PIXImus, GE LUNAR Corporation), and body weightchange was determined by weighing the animals periodically for 37 days.The results at the end of this comparative study was that ActRIIB-Fc(E28W) (SEQ ID NO: 21) showed an increase of 24% in lean muscle mass and25% in increase of body weight compared with an increase of 25% in leanmuscle mass and 20% increase in body weight for svActRIIB-Fc (E28W,S44T) (SEQ ID NO: 10), compared with an increase of 5% lean muscle massand 9% increase body weight for the control group.

Therefore, it can be seen that svActRIIB-Fc (E28W, S44T) retainscomparable in vivo efficacy compared with ActRIIB-Fc (E28W) while havingimproved manufacturability characteristics.

Example 4 Improved Manufacturability with Modified Hinge Linkers

Additional linkers and modified hinge regions were constructed to testfor further improvement of protein expression and manufacturability ofthe stabilized ActRIIB (E28W, S44T) polypeptides. Modified linker/hingesequences based on modifications of hinge linker #1 were generated usingoverlap extension PRC mutagenesis methods, according to Mikaelian etal., Methods in Molecular Biology, 57, 193-202 (1996), and well knownmethodology.

The modified hinge linkers designed to perform well with IgG2 Fc fusionsare hinge linker #2-7 set forth below (in comparison to hinge linker #1sequences).

hinge linker #1 polynucleotide (SEQ ID NO: 26)ggagggggaggatctgtcgagtgcccaccgtgccca. hinge linker #1 polypeptide(SEQ ID NO: 27) GGGGSVECPPCP hinge linker #2 polynucleotide(SEQ ID NO: 37) ggagggggaggatctgagcgcaaatgttgtgtcgagtgcccaccgtgchinge linker #2 peptide (SEQ ID NO: 38) GGGGSERKCCVECPPChinge linker #3 polynucleotide (SEQ ID NO: 39)ggagggggaggatctggtggaggtggttcaggtccaccgtgc hinge linker #3 peptide(SEQ ID NO: 40) GGGGSGGGGSGPPC hinge linker #4 polynucleotide(SEQ ID NO: 41) ggagggggaggatctggtggaggtggttcaggtccaccgggahinge linker #4 peptide (SEQ ID NO: 42) GGGGSGGGGSGPPGhinge linker #5 polynucleotide (SEQ ID NO: 43)ggagggggaggatctgagcgcaaatgtccaccttgtgtcgagtgc ccaccgtgchinge linker #5 peptide (SEQ ID NO: 44) GGGGSERKCPPCVECPPChinge linker #6 peptide (SEQ ID NO: 45) GPASGGPASGPPCPhinge linker #7 peptide (SEQ ID NO: 46) GPASGGPASGCPPCVECPPCP

The following hinge linkers #8 to #10 below were designed to performwell with an IgG1Fc (SEQ ID NO: 23) or the modified IgG1Fc given below(SEQ ID NO: 47 below).

modified IgG1 Fc (SEQ ID NO: 47)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hinge linker #8 peptide(SEQ ID NO: 48) GGGGSVDKTHTCPPCP hinge linker #9 peptide (SEQ ID NO: 49)GGGGSVDKTHTGPPCP hinge linker #10 peptide (SEQ ID NO: 50)GGGGSGGGGSVDKTHTGPPCP

Testing of modified hinge linker sequences with svActRIIB-Fc (28W, S44T)was performed as follows. Polynucleotides encoding svActRIIB (E28W,S44T) (SEQ ID NO: 5), polynucleotides encoding the modified hingelinkers shown above, and polynucleotides encoding IgG2 Fc (SEQ ID NO:22) or polynucleotides encoding IgG1 Fc (SEQ ID NO: 23) or modified IgG1Fc (SEQ ID NO: 47) were subcloned into vectors as described in Example 1and expressed using the transient 293-6E expression system as describedin Example 1, except for the following changes: F17 media (Invitrogen)supplemented with 1.1 mg/ml Pluronic, 6 mM L-glutamine and 25 μg/mlgeneticin (Invitrogen) was used in place of Freestyle 293 medium asdescribed in Durocher et al., Nucleic Acids Research 30, No. 3, e9(2002)). The cultures were grown for seven days at 37° C. aftertransfection. Aliquots were centrifuged to remove cells, and thesupernatant was mixed with loading buffer before being heated and loadedonto a 4-20% tris-glycine gel for analysis by Western Blot. After theprotein was transferred to a nitrocellulose membrane, samples wereprobed with a hydrogen peroxidase conjugated anti-human Fc antibody(Pierce #31423) at a dilution of 1:1000.

Protein purification was performed using the following procedure.Approximately 0.25 L of the conditioned media containing thesvActRIIB-Fc variants were concentrated using a 5 ft² 10 K membranetangential flow filter. The concentrated material was applied to a 5 mLProtein A High Performance Column™ (GE Heathcare) which had beenequilibrated with PBS (Dulbecco's with no magnesium chloride or calciumchloride). After washing the column with the equilibration buffer untilthe absorbance at 280 nm (OD₂₈₀) was less than 0.1, the bound proteinwas eluted with 0.1 M glycine-HCl, pH 2.7, and immediately neutralizedwith 1 M Tris-HCl, pH 8.5.

The portion of aggregate in percent and the portion of half molecule inpercent were determined by the following method. Denaturing sizeexclusion chromatography experiments were performed by injecting a 50 μlaliquot of each sample onto an HPLC system with two size exclusioncolumns (TOSOHAAS G3000swxl) in tandem. The mobile phase contains 5 MGuHCl in phosphate buffered saline (PBS). All samples were diluted to 1mg/mL in PBS with 7 M GuHCl. The portion of aggregate in percent isdetermined from the total peak areas of the peaks eluted before the mainpeak, whereas the portion of half-molecule in percent is determined fromthe total peak areas of the peaks eluted after the main peak. Thehalf-molecule are believed to represent inactive half-molecules.

Aggregate and half-molecule distribution of svActRIIB-Fc (E28W, S44T)with the various hinge linkers are set forth in the following table.

Hinge linker % % half sequence aggregate molecule GGGGSVECPPC  0.6315.12 (SEQ ID NO: 27) GGGGSERKCCVECPPC 15.01  7.19 (SEQ ID NO: 38)GGGGSGGGGSGPPC  0.56  3.83 (SEQ ID NO: 40) GGGGSGGGGSGPPG  0.00 99.03(SEQ ID NO: 42) GGGGSERKCPPCVECPPC  1.09  3.81 (SEQ ID NO: 44)

Thus certain linkers may improve manufacturability of the stabilizedActRIIB-Fc (E28W, S44T) according to these preliminary tests by reducingthe percentage of inactive half-molecules produced.

The table below identifies the sequences as listed in the sequencelisting.

SEQ ID NO Description 1 ActRIIB extracellular domain, polynucleotide 2ActRIIB extracellular domain, polypeptide 3 svActRIIB (E28W, S44T)polynucleotide with signal sequence 4 svActRIIB (E28W, S44T) polypeptidewith signal sequence 5 svActRIIB (E28W, S44T) polynucleotide withoutsignal sequence 6 svActRIIB (E28W, S44T) polypeptide without signalsequence 7 svActRIIB-Fc (E28W, S44T) polynucleotide with signal sequence8 svActRIIB-Fc (E28W, S44T) polypeptide with signal sequence 9svActRIIB-Fc (E28W, S44T) polynucleotide without signal sequence 10svActRIIB-Fc (E28W, S44T) polypeptide without signal sequence 11svActRIIB (E28Y, S44T) polynucleotide with signal sequence 12 svActRIIB(E28Y, S44T) polypeptide with signal sequence 13 svActRIIB (E28Y, S44T)polynucleotide without signal sequence 14 svActRIIB (E28Y, S44T)polypeptide without signal sequence 15 svActRIIB-Fc (E28Y, S44T)polynucleotide with signal sequence 16 svActRIIB-Fc (E28Y, S44T)polypeptide with signal sequence 17 svActRIIB-Fc (E28Y, S44T)polynucleotide without signal sequence 18 svActRIIB-Fc (E28Y, S44T)polypeptide without signal sequence 19 ActRIIB (E28W) polypeptide,without signal sequence 20 ActRIIB-Fc (E28W) polynucleotide, withoutsignal sequence 21 ActRIIB-Fc (E28W) polypeptide, without signalsequence 22 IgG2Fc polypeptide sequence 23 IgG1Fc polypeptide sequence24 IgG4 Fc polypeptide sequence 25 Linker amino acid sequence 26 Hingelinker #1 polynucleotide sequence 27 Hinge linker #1 peptide sequence 28Hinge region IgG2 29 Hinge region IgG1 30 Hinge region IgG4 31Alternative signal sequence, polypeptide 32 Signal sequence, polypeptide33 Wild type ActRIIB accession NP_001097 34 Activin polypeptide sequence35 Myostatin polypeptide sequence 36 GDF-11 polypeptide sequence 37Hinge linker sequence #2 polynucleotide 38 Hinge linker sequence #2peptide 39 Hinge linker sequence #3 polynucleotide 40 Hinge linkersequence #3 peptide 41 Hinge linker sequence #4 polynucleotide 42 Hingelinker sequence #4 peptide 43 Hinge linker sequence #5 polynucleotide 44Hinge linker sequence #5 peptide 45 Hinge linker sequence #6 peptide 46Hinge linker sequence #7 peptide 47 Modified IgG1 Fc polypeptidesequence 48 Hinge linker sequence #8 peptide 49 Hinge linker sequence #9peptide 50 Hinge linker sequence #10 peptide

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

What is claimed is:
 1. An isolated protein comprising a stabilizedactivin IIB receptor polypeptide (svActRIIB) wherein said polypeptide isselected from the group consisting of: (a) a polypeptide consisting ofthe sequence set forth in SEQ ID NO: 2, except for a single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected fromthe group consisting of W and Y, and the substitution at position 44 isT; (b) a polypeptide consisting of the sequence set forth in amino acids19 through 134 of SEQ ID NO: 2, except for a single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected fromthe group consisting of W and Y, and the substitution at position 44 isT; (c) a polypeptide consisting of the sequence set forth in amino acids23 through 134 of SEQ ID NO: 2, except for a single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected fromthe group consisting of W and Y, and the substitution at position 44 isT; (d) a polypeptide consisting of the sequence set forth in amino acids25 through 134 of SEQ ID NO: 2, except for a single amino acidsubstitution at position 28, and a single amino acid substitution atposition 44, wherein the substitution at position 28 is selected fromthe group consisting of W and Y, and the substitution at position 44 isT; (e) a polypeptide having at least 80% sequence identity to any one of(a) through (d), except for a single amino acid substitution at position28, and a single amino acid substitution at position 44, wherein thesubstitution at position 28 is selected from the group consisting of Wand Y, and the substitution at position 44 is T, wherein the polypeptideis capable of binding myostatin, activin A, or GDF-11.
 2. An isolatedprotein comprising a stabilized activin IIB receptor polypeptide,wherein said polypeptide is selected from the group consisting of: (a) apolypeptide consisting of the sequence set forth in the group consistingof SEQ ID NO: 4, 6, 12 and 14; (b) a polypeptide having at least 90%sequence identity to (a), wherein the polypeptide has a W or a Y atposition 28 and a T at position 44, wherein the polypeptide is capableof binding myostatin, activin A, or GDF-11, and (c) polypeptides havingat least 95% sequence identity to (a), wherein the polypeptide has a Wor a Y at position 28 and a T at position 44, wherein the polypeptide iscapable of binding myostatin, activin A, or GDF-11.
 3. The protein ofclaim 2, wherein the polypeptide is connected to at least oneheterologous protein.
 4. The protein of claim 3, wherein theheterologous polypeptide is an IgG Fc domain.
 5. The protein of claim 3,wherein the heterologous polypeptide is connected to the polypeptide bya linker sequence.
 6. The protein of claim 5, wherein the linker isselected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO:
 50. 7. Theprotein of claim 4, wherein the protein comprises a polypeptide selectedfrom the group consisting of: (a) a polypeptide consisting of thesequence set forth in the group consisting of SEQ ID NO: 8, 10, 16 and18; (b) a polypeptide having at least 90% sequence identity to (a),wherein the polypeptide has a W or a Y at position 28 and a T atposition 44, wherein the polypeptide is capable of binding myostatin,activin A, or GDF-11, and (c) polypeptides having at least 95% sequenceidentity to (a), wherein the polypeptide has a W or a Y at position 28and a T at position 44, wherein the polypeptide is capable of bindingmyostatin, activin A, or GDF-11.
 8. An isolated nucleic acid moleculecomprising a polynucleotide encoding the polypeptide of claim
 1. 9. Anisolated nucleic acid molecule comprising a polynucleotide selected fromthe group consisting of: (a) a polynucleotide encoding a polypeptideconsisting of the sequence set forth in the group consisting of SEQ IDNO: 4, 6, 12, and 14; (b) a polynucleotide encoding a polypeptide havingat least 90% sequence identity to (a), wherein the polypeptide has a Wor a Y at position 28 and a T at position 44; and wherein thepolypeptide is capable of binding myostatin, activin A, or GDF-11; and(c) a polynucleotide encoding a polypeptide having at least 95% sequenceidentity to (a), wherein the polypeptide has a W or a Y at position 28and a T at position 44, and wherein the polypeptide is capable ofbinding myostatin, activin A, or GDF-11.
 10. The nucleic acid moleculeof claim 9, wherein the polynucleotide has the sequence selected fromthe group consisting of SEQ ID NO: 3, 5, 11 and 13 or its complement.11. The nucleic acid molecule of claim 9, wherein the polynucleotidefurther comprises polynucleotides encoding at least one heterologousprotein.
 12. The nucleic acid molecule of claim 11, comprising apolynucleotide selected from the group consisting of: (a) apolynucleotide encoding a polypeptide consisting of the sequence setforth in the group consisting of SEQ ID NO: 8, 10, 16, and 18; (b) apolynucleotide encoding a polypeptide having at least 90% sequenceidentity to (a), wherein the polypeptide has a W or a Y at position 28and a T at position 44, and wherein the polypeptide is capable ofbinding myostatin, activin A, or GDF-11; and (c) a polynucleotideencoding a polypeptide having at least 95% sequence identity to (a),wherein the polypeptide has a W or a Y at position 28 and a T atposition 44, and wherein the polypeptide is capable of bindingmyostatin, activin A, or GDF-11.
 13. The nucleic acid molecule of claim12, wherein the polynucleotide has the sequence selected from the groupconsisting of SEQ ID NO: 7, 9, 15 and 17, or it complement.
 14. Thenucleic acid molecule of claim 9, wherein the polynucleotide is operablylinked to a transcriptional or translational regulatory sequence.
 15. Arecombinant vector comprising the nucleic acid molecule of claim
 9. 16.A host cell comprising the recombinant vector of claim
 15. 17. The hostcell of claim 16, wherein the host cell is a mammalian cell.
 18. Amethod of producing a svActRIIB protein comprising culturing the hostcell of claim 16 under conditions promoting the expression of theprotein, and recovering the protein.
 19. A pharmaceutical compositioncomprising an effective amount of the protein of claim 2 in admixturewith a pharmaceutically acceptable carrier.
 20. A method of inhibitingmyostatin activity or activin activity in a subject in need of suchtreatment, a method of treating a disease in which activin isoverexpressed in a subject in need of such treatment, or a method oftreating a muscle wasting or metabolic or activin related disorder in asubject in need of such a treatment comprising administering atherapeutically effective amount of the composition of claim 19 to thesubject.